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Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina.

Chen GE, Hitchcock A, Jackson PJ, Chaudhuri RR, Dickman MJ, Hunter CN, Canniffe DP - J. Bacteriol. (2016)

Bottom Line: Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB.Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed.Carrying a reduced C-8 group may be of particular importance to organisms containing chlorophyll d Plant genomes also contain orthologs of both of these genes; thus, the bacterial progenitor of the chloroplast may also have contained both bciA and bciB.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Detection of transcripts of bciA and bciB in A. marina by RT-PCR. Reactions for bciA and bciB, along with the rnpB housekeeping control, were performed with the inclusion (+) and omission (−) of reverse transcriptase to ensure that samples were not contaminated by genomic DNA.
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Figure 4: Detection of transcripts of bciA and bciB in A. marina by RT-PCR. Reactions for bciA and bciB, along with the rnpB housekeeping control, were performed with the inclusion (+) and omission (−) of reverse transcriptase to ensure that samples were not contaminated by genomic DNA.

Mentions: In order to determine which of the 8VRs A. marina utilizes for Chl biosynthesis or whether both proteins are employed, transcription of bciA and bciB was checked by RT-PCR, and the presence of the cognate proteins was determined by mass spectrometry. Total A. marina RNA was isolated from a culture at mid-exponential growth phase, treated with DNase to remove any genomic DNA, and used as the template for one-step RT-PCR in which cDNA synthesis and PCR amplification were performed in a single reaction. The housekeeping gene rnpB, encoding the RNA subunit of RNase P, was included as positive control. The amplicon generated by RT-PCR displayed a single band with the expected sizes for all the three genes when analyzed by agarose gel electrophoresis (Fig. 4): 140 bp for bciA, 142 bp for bciB, and 106 bp for rnpB. The absence of bands in the no-RT controls eliminated the possibility of genomic DNA contamination. Therefore, we can conclude that both nmrA and frhB are actively transcribed under the conditions tested in A. marina. Mass spectrometry analysis was performed to verify the presence of NmrA and FrhB proteins in A. marina. Proteins extracted from an A. marina whole-cell lysate were treated with a combination of endoproteinase LysC and trypsin to generate peptide fragments which were then analyzed by nano-liquid chromatography (LC)-MS/MS. Mass spectra, consisting of both peptide ion masses and their product ion profiles, were used as inputs for searching against the A. marina reference proteome database. In total, 1,470 proteins were identified, including both NmrA and FrhB, as shown in Table 2.


Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina.

Chen GE, Hitchcock A, Jackson PJ, Chaudhuri RR, Dickman MJ, Hunter CN, Canniffe DP - J. Bacteriol. (2016)

Detection of transcripts of bciA and bciB in A. marina by RT-PCR. Reactions for bciA and bciB, along with the rnpB housekeeping control, were performed with the inclusion (+) and omission (−) of reverse transcriptase to ensure that samples were not contaminated by genomic DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836224&req=5

Figure 4: Detection of transcripts of bciA and bciB in A. marina by RT-PCR. Reactions for bciA and bciB, along with the rnpB housekeeping control, were performed with the inclusion (+) and omission (−) of reverse transcriptase to ensure that samples were not contaminated by genomic DNA.
Mentions: In order to determine which of the 8VRs A. marina utilizes for Chl biosynthesis or whether both proteins are employed, transcription of bciA and bciB was checked by RT-PCR, and the presence of the cognate proteins was determined by mass spectrometry. Total A. marina RNA was isolated from a culture at mid-exponential growth phase, treated with DNase to remove any genomic DNA, and used as the template for one-step RT-PCR in which cDNA synthesis and PCR amplification were performed in a single reaction. The housekeeping gene rnpB, encoding the RNA subunit of RNase P, was included as positive control. The amplicon generated by RT-PCR displayed a single band with the expected sizes for all the three genes when analyzed by agarose gel electrophoresis (Fig. 4): 140 bp for bciA, 142 bp for bciB, and 106 bp for rnpB. The absence of bands in the no-RT controls eliminated the possibility of genomic DNA contamination. Therefore, we can conclude that both nmrA and frhB are actively transcribed under the conditions tested in A. marina. Mass spectrometry analysis was performed to verify the presence of NmrA and FrhB proteins in A. marina. Proteins extracted from an A. marina whole-cell lysate were treated with a combination of endoproteinase LysC and trypsin to generate peptide fragments which were then analyzed by nano-liquid chromatography (LC)-MS/MS. Mass spectra, consisting of both peptide ion masses and their product ion profiles, were used as inputs for searching against the A. marina reference proteome database. In total, 1,470 proteins were identified, including both NmrA and FrhB, as shown in Table 2.

Bottom Line: Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB.Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed.Carrying a reduced C-8 group may be of particular importance to organisms containing chlorophyll d Plant genomes also contain orthologs of both of these genes; thus, the bacterial progenitor of the chloroplast may also have contained both bciA and bciB.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom.

No MeSH data available.


Related in: MedlinePlus